The present invention relates generally to materials and methods for adsorbing macromolecules such as enzymes and other proteins, as might be particularly useful in enzyme immobilization and protein separation.
Enzymes and other proteins are highly useful materials and are used in numerous ways. For example, in order to capitalize on the ability of enzymes to catalyze almost any type of chemical reaction with extraordinary specificity and efficacy, one significant use for enzymes is as catalysts in industrial-scale chemical synthesis. Because enzymes are difficult to recover and are highly susceptible to denaturation by chemical or physical factors such as pH extremes, temperature and/or the presence of organic materials, it is important that the enzymes be immobilized, or compartmentalized, to enhance their use in bulk chemical synthesis.
Previous attempts to immobilize enzymes to permit their use in industrial reactions have not met with success. One prior approach involved the use of microcapsules to create an outer barrier for protecting the active enzyme therein. The concept of microencapsulation is ill-suited to immobilize enzymes because of insufficient transport kinetics for permitting diffusion of the enzyme in and out of the microcapsule. As a result, the wall or barrier provided by the microcapsule does not provide the enzyme with sufficient exposure to the medium in which it is supposed to act thereby resulting in a loss of enzymatic activity. Other prior approaches included alginate beads, cross-linked polyurethane, starch particles and polyacrylamide gels. Not only are the procedures for forming these materials quite complex, but the materials are soft gels and are therefore not mechanically strong.
A more recent approach for immobilizing enzymes has involved the use of coacervates, which are aggregates of colloidal droplets arising via aggregation primarily through the force of electrostatic attraction. However, the use of coacervates to incorporate enzymes, while not using a capsule, still has not been fully satisfactory. For example, coacervates tend to aggregate and collect into larger particles and are therefore subject to instability. Whereas it is normally desired that the coacervate droplets have a diameter of approximately 1 micrometer, these droplets can undesirably combine to become 10 micrometers or more in diameter. These larger droplets result in a proportionately smaller surface area in which the enzymes can act, and consequently, do not optimize the reaction rates. In addition, the coacervates themselves are somewhat fragile and are not capable of being subjected to, for example, flow-through reactors. Coacervates also do not constitute a versatile design and require low-speed centrifugation for removal.
Another area in which proteins must be adapted for use relates to protein purification or separation in which a target protein is separated from one or more other components such as other proteins or nucleic acids. Prior attempts to purify proteins have involved a large number of steps, often 17 or more steps, to ultimately purify and separate a target protein. These numerous steps can include gross filtration, centrifugation, settling, salting out, two-phase extraction, one or more chromatography steps, or other steps. For example, a cell crush may initially be purified to remove some components, such as lipids, sugars, particulates, and cell debris. However, removal of a target protein from other proteins and nucleic acids is still time-consuming, expensive and complex because of the requirement of numerous steps and can be diluting. Strictly by way of example, recombinant DNA techniques that utilize yeast or bacteria in making human insulin result in a cell crush that requires a number of purification steps, ranging from coarse filtration to very detailed fine tuned chromatography that greatly contribute to the expense of the final product.
From the foregoing, it will be appreciated that there exists a need in the enzyme immobilization art for an enzyme substrate that supplies sufficient mechanical strength while permitting the enzyme to be highly exposed to the medium in which it is supposed to act. There also exists a need for such an enzyme substrate where there is no barrier or wall that requires diffusion in and out of the barrier that causes a loss of enzymatic activity. It will also be appreciated that there exists a need in the protein purification art for methods and materials for purifying and separating a target protein from other components present in a medium that does not require numerous steps.